The level of certain analytes in blood and other body fluids can predict disease or risk thereof. For example, cholesterol in blood is a significant indicator of risk of coronary heart disease. “Total cholesterol” includes low density lipoproteins (LDL), very low density lipoproteins (VLDL) and high density lipoproteins (HDL). It is well established from epidemiological and clinical studies that there is a positive correlation between levels of LDL and VLDL cholesterol (“bad” cholesterol) and coronary heart disease and a negative correlation between levels of HDL cholesterol (“good” cholesterol) and coronary heart disease. The level of total cholesterol in blood, which is a measure of the sum total of HDL, LDL, VLDL and chylomicrons, is not generally regarded as an adequate indicator of the risk of coronary heart disease because the overall level of total cholesterol does not reveal the relative proportions of HDL, LDL and VLDL. To better assess the risk of heart disease, it is desirable to determine the amount of HDL, LDL and triglycerides in addition to total cholesterol. Physicians commonly order what is referred to in the art as a “full lipid panel” for their patients. A lipid panel includes concentration of total cholesterol, HDL cholesterol, LDL cholesterol and triglycerides.
There are known test devices that can determine the level of multiple individual analytes, but they undesirably require a separate test strip and a separate fluid sample for each analyte to be determined. If the fluid sample be whole blood, the battery of tests undesirably requires taking multiple samples of blood, or taking an undesirably large single sample and then separately depositing portions thereof onto individual test strips.
For example, U.S. Pat. No. 5,597,532 discloses an excellent apparatus for optoelectronic evaluation of test paper strips for use in the detection of certain analytes in blood or other body fluids. The test strip comprises an elongated plastic part including a hinged portion to allow a first portion to be folded over a second portion. A series of layers of test strips are disposed between the folded over portions of the test strip. The method involves providing a separately colored strip and corresponding memory module for each test. For example, total cholesterol strips and modules may be colored red, whereas glucose strips and modules may be colored yellow, and so forth. However, a separate sample must be used and a separate test conducted for each analyte for which concentration is to be determined.
One problem in designing a multiple analyte test strip lies with blood cell separation, in that most dry phase test strips separate red blood cells by a lateral flow scheme. For example, U.S. Pat. No. 4,816,224 (Rittersdorf et al.) discloses a glass fiber matrix for blood cell separation in which a blood sample is placed on the matrix and lateral movement through the length of the matrix ensues. Red blood cells and plasma both migrate laterally across the fiber matrix, but the red blood cells migrate at a slower rate than plasma. Further, some hemolysis eventually occurs in the glass fiber layer. Further, many commercially available lateral flow devices are configured such that the reaction layer is not brought into fluid-conveying contact with the glass fiber layer until the glass fiber layer is completely filled with plasma. This happens at a predetermined and exact time after an adequate amount of plasma, but not red blood cells, has migrated to a designated location on the glass fiber layer.
Further, determining concentrations from whole blood of certain analytes, e.g., HDL, requires multiple process steps, and the prior art known to applicant teaches that many or all of these process steps are to be conducted via lateral flow schemes. For example, U.S. Pat. No. 5,426,030 (Rittersdorf et al.) and its progeny disclose test strips for precipitation and separation of non-HDL cholesterol from HDL cholesterols in a plasma sample. This separation technology involves two layers in contact with one another. The first layer is made from a hydrophilic glass fiber layer impregnated with a precipitating agent that precipitates non-HDLs but not HDLs. The second layer is preferably a mesh glass fiber layer with fibers of a diameter of 0.2 to 10.0 μm that acts as a transport medium. Precipitation of non-HDL cholesterols occurs in the first layer and separation of the non-HDL precipitants from the plasma occurs as the plasma having precipitated non-HDLs migrates across the second layer. Again, however, the separation step is understood by applicant to be a chromatographic technique which applicants believe may limit the versatility of the test. For example, it may be difficult to design and implement a dry phase test strip that utilizes two lateral flow operations, one to separate blood and the other to precipitate and retain non-HDLs.
One dry phase test strip device known to applicants for measuring multiple analytes in a single whole blood sample is disclosed in U.S. Pat. No. 5,213,965 (Jones). This device measures concentration of HDL cholesterol and other analytes from a whole blood sample, but the device is rather complex. The device includes a well in which the whole blood sample is deposited and then drawn through a capillary to a sieving pad made of fibrous material. The sieving pad achieves initial separation of blood cells from plasma on the basis of the blood cell's slower migration rate therethrough. The sieving pad is covered with a microporous membrane which further filters blood cells. Covering the microporous membrane is a reagent reservoir membrane containing precipitating agents for non-HDLs on one side thereof. On the other side of the reagent reservoir, there are no precipitating agents.
On top of and extending laterally beyond the reagent reservoir is an elongate matrix which distributes the sample laterally after it leaves the reservoir. Finally, one or more test pads are positioned above and biased apart from the elongate matrix. Plasma exits the filtering membrane and enters the reagent reservoir where non-HDLs are precipitated on one side thereof and then flow from the reservoir and migrate laterally through one side of the elongate matrix. Similarly, plasma that enters the other side of the reagent reservoir encounters no precipitating agents, and this plasma exits the side of the elongate matrix opposite the side the plasma containing precipitated nonHDLs exits. At a desired time, the test pads can be depressed so they are in fluid communication with the elongate matrix. The test pads that contact one side of the elongate matrix measure concentration of HDL, whereas the test pads that contact the opposite side of the elongate matrix measure total cholesterol.
Undesirably, the device disclosed by the '965 patent relies upon not one, but two, separate chromatographic operations or lateral flow schemes, the first being blood separation in the sieving pad, and the second being separation of non-HDLs across the elongate matrix. Further, the device disclosed by the '965 patent is undesirably complex. For example, it requires a well, a capillary tube, two layers to separate blood, and two layers to precipitate and then separate non-HDLs. Finally, the test pads must be kept spaced apart from the elongate matrix until the entire operation is properly timed, whereupon the test plate having the test pads thereon can be depressed against the elongate matrix. The test pads are held against the elongate matrix for a predetermined time, then removed, so as to tightly control the volume of sample received by the test pads. Of course, depressing and then lifting the test pad requires process steps and associated structure to carry out those steps.
It is desirable to avoid the lateral flow schemes, chromatographic operations, complex devices and the delicate timing operations that are required by the prior art disclosed above. Generally, it is desirable to provide a test strip for measuring concentration of multiple analytes from a single sample that is more reliable, economical, easier to use and less prone to error than the prior art devices discussed above.